PROJECT SUMMARY/ABSTRACT We propose to develop a mechanism for effective targeting of neoantigens in salivary gland cancers (SGCs), which are understudied, treatment-resistant malignancies. This is the first step toward our long-term goal of employing immunotherapy as a curative treatment for patients with SGC. Currently, most recurrent and metastatic SGCs are incurable, and effective therapy is lacking. The efficacy of immune checkpoint inhibitors (ICIs) in SGCs has been largely unexplored, though preliminary data suggest modest response rates. Drawing upon our understanding of other cancer types, we reason that neoantigens and other genetic and immunologic features of SGCs may reflect their likelihood of response to ICI, and that such knowledge will reveal tumor vulnerabilities and nominate the most promising immunotherapeutic strategies for clinical investigation. In our preliminary data, we show that SGC histologies differ greatly in mutational/neoantigen load and immune infiltration. Therefore, it is unlikely that one clinical approach will be effective across histologies. We seek to identify and test neoantigen and immunologic vulnerabilities in four aggressive types of SGC: high-grade adenocarcinoma and adenoid cystic, salivary duct, and myoepthielial carcinomas. We will first define the mechanisms of immunogenicity and immune escape in these salivary cancers (Aim #1) through integrated analyses of exome and RNA sequencing and multiplex immunohistochemistry of clinical tumor samples, and through mass cytometry of tumor-infiltrating lymphocytes. Of particular interest is the identification of fusion- associated neoantigens that are shared across multiple SGC patients. We will then identify genetic and immunologic determinants of SGC response to ICI (Aim #2), taking advantage of biopsies and blood samples collected as part of our ongoing Phase II clinical trial of combined T cell checkpoint inhibitor therapy (nivolumab plus ipilimumab). We will have access to serial tissue and blood samples from 64 SGC patients treated with this combination, enabling exome and RNA sequencing and multiplex immunohistochemistry in tumor samples and T cell receptor (TCR) sequencing in blood. We will test whether neoantigen load and immune infiltration correlate with response to treatment; we will also test other genetic and immune factors for correlation with outcome in exploratory analyses. In Aim #3, we will focus on adenoid cystic carcinoma (ACC), the most common metastatic SGC histology. Most ACCs are driven by a MYB-NFIB gene fusion, which we have found can generate immunogenic neoantigens. In patient samples, we will identify fusion-associated immunogenic neoantigens, then generate TCR-engineered T cells targeting shared neoantigens, and test their efficacy in humanized mouse models of ACC. Our well-established multi-disciplinary team of SGC investigators will carry out this research, which will help identify targets for immunotherapy, identify new biomarkers, and establish the feasibility of targeting fusion gene?associated neoantigens with engineered T cells.